Hydrolytic inactivation of a breast carcinoma cell-derived serpin by human stromelysin-3

J Biol Chem. 1994 Oct 14;269(41):25849-55.


To elucidate structure-function relationships of stromelysin-3, a putative matrix metalloproteinase originally identified at the tumor-stromal cell interface in breast carcinomas, the human cDNA was expressed in mammalian cells, and its products were isolated and characterized. In stably transfected cells, stromelysin-3 was recovered as a complex mixture of species ranging in size from approximately 20 to 65 kDa. Among these products, a major 45-kDa species with an N terminus of Phe98 and an intact C-terminal domain was identified as a true endopeptidase on the basis of its ability to cleave the bait region of alpha 2-macroglobulin between Phe684 and Tyr685, a site identical to that recognized by stromelysin-1. However, unlike stromelysin-1 or other members of the matrix metalloproteinase family, the mature form of stromelysin-3 was unable to hydrolyze a range of extracellular matrix molecules associated with either the basement membrane or interstitium. To probe for alternate substrates among tumor cell-derived products, purified stromelysin-3 was incubated with [35S]methionine-labeled medium conditioned by the breast cancer cell line, MCF-7. Under these conditions, a single, tumor cell-derived protein was hydrolyzed as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Following anion-exchange chromatography and preparative gel electrophoresis, the stromelysin-3 substrate was identified by N-terminal sequencing as the serine proteinase inhibitor, alpha 1-proteinase inhibitor. Further studies demonstrated that stromelysin-3 rapidly destroyed the antiproteolytic function of alpha 1-proteinase inhibitor by cleaving the antiproteinase at a distinct site between Ala350 and Met351 within the reactive-site loop. Together, these data not only demonstrate that human stromelysin-3 acts as a powerful endopeptidase with a restricted substrate specificity distinct from all other matrix metalloproteinases, but also serve to identify serine proteinase inhibitors as potential physiologic targets at sites of extracellular matrix remodeling.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Breast Neoplasms / enzymology*
  • Carcinoma / enzymology*
  • Caseins / metabolism
  • Extracellular Matrix Proteins / classification
  • Female
  • Humans
  • Hydrolysis
  • Leukocyte Elastase*
  • Matrix Metalloproteinase 11
  • Metalloendopeptidases / classification
  • Metalloendopeptidases / genetics
  • Metalloendopeptidases / metabolism*
  • Molecular Sequence Data
  • Neoplasm Proteins / biosynthesis
  • Neoplasm Proteins / genetics
  • Neoplasm Proteins / pharmacology*
  • Pancreatic Elastase / metabolism
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / pharmacology
  • Serpins / drug effects
  • Serpins / metabolism*
  • Structure-Activity Relationship
  • Substrate Specificity
  • alpha 1-Antitrypsin / metabolism
  • alpha-Macroglobulins / metabolism


  • Caseins
  • Extracellular Matrix Proteins
  • Neoplasm Proteins
  • Recombinant Proteins
  • Serpins
  • alpha 1-Antitrypsin
  • alpha 1-antitrypsin-leukocyte elastase complex
  • alpha-Macroglobulins
  • Pancreatic Elastase
  • Leukocyte Elastase
  • Matrix Metalloproteinase 11
  • Metalloendopeptidases